It has been known that living cells of bacteria belonging to hemolytic streptococci have a certain anti-cancer activity. However, because the activity is very unstable, many approaches have been tried to increase its stability. U.S. Pat. Nos. 3,477,914, 3,729,554 and 3,632,747 and Swiss Pat. No. 543,227 report various practicable methods wherein living cells of the bacteria are suspended in a saline solution containing an antibiotic such as penicillin, cephalosporin, cycloserine or the like, heated at a temperature ranging from 37.degree. to 45.degree. C. and, after adding a suitable stabilizer to the suspension, lyophilized to give dried preparations.
Although these preparations exhibit strong anti-tumor activities, they have defects for pharmaceutical preparation such that they induce pain in the place where administered to a patient, have inflaming property and induce transient fever in a patient upon administration.
On the other hand, various methods of extracting anti-tumor substances from the cells of bacteria belonging to hemolytic streptococci have been studied. Japanese Patent Publication No. 1647/63 reported a method which comprises disrupting the cells to recover the supernatant, adding an organic solvent to the supernatant and collecting the precipitating antibiotic substances. Further, U.S. Pat. No. 3,810,819 disclosed a method which comprises disrupting or lysing the cells to recover the supernatant and collecting active substances as a fraction containing 50-80% saturated ammonium sulfate solution. These anti-tumor substances recovered from the water-soluble fractions are comprised mainly of proteins and are extremely unstable against heat and are considerably lower in anti-tumor activity than a preparation containing cells. In addition, they have a high inflaming property and there is relatively high possibility to induce fever or pain in a patient upon administration.
Also, a method has been known from U.S. Pat. No. 3,786,141, which comprises having a lytic enzyme act on cells of the bacteria belonging to hemolytic streptococci and recovering water-insoluble active substances from the lysed solution. This type of substance comprises mainly protoplasmic membrane of the cells, and has essentially no inflaming property, pyrogenic activity or pain-inducing property. However, the anti-tumor activity of the object substance is unstable against heat.
During the study, the inventors have found that substances which were obtained by physically disrupting cells of bacteria belonging to hemolytic streptococci to recover a water-insoluble fraction mainly comprising cell wall components and by treating the fraction with a nuclease or protease, have low inflaming and pain-inducing properties and a strong anti-tumor activity with good stability against heat. The inventors have continued their studies based on such facts to complete this invention.